Protein stabilization formulations

ABSTRACT

The present invention is directed to stabilizing Bone Morphogenetic Protein in various lyophilized formulations and compositions. The present invention comprises formulations primarily including trehalose as an excipient for lyophilized compositions and their subsequent storage and reconstitution, and can also optionally include other excipients, including buffers and surfactants.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 13/089,683entitled “PROTEIN STABILIZATION FORMULATIONS”, filed on Apr. 19, 2011,now U.S. Pat. No. 8,435,943 which is a divisional of U.S. applicationSer. No. 11/950,127 entitled “PROTEIN STABILIZATION PORMULATIONS”, filedon Dec. 4, 2007, now U.S. Pat. No. 7,956,028 which is a non provisionalof provisional filing, U.S. App. Pat. No. U.S. App. Pat. No. 60/870,032entitled “PROTEIN STABILIZATION FORMULATIONS,” filed on Dec. 14, 2006.

FIELD OF THE INVENTION

The present invention is directed toward formulations and methods forstabilizing bone morphogenetic proteins (BMP's) and the closely relatedgrowth and differentiation factors (GDF's) during processing, storage,and reconstitution. More particularly, the present invention relates toformulations comprised of trehalose and other excipients to protectrhGDF-5 during lyophilization, storage, and reconstitution, includingvarious substrates used as a vehicle to deliver rhGDF-5. Additionally,the present invention includes methods for preparing and using suchformulations to treat various musculoskeletal defects and conditions.

BACKGROUND OF THE INVENTION

Biological molecules (biomolecules) have three-dimensional structure orconformation, and rely on this structure for their biological activityand properties. Examples of such biomolecules include deoxyribonucleicacid (DNA), ribonucleic acid (RNA), and proteins. These biomolecules areessential for life, and represent therapeutic agents and targets intreating various medical diseases and conditions. Proteins represent abroad class of biomolecules. Different classes of proteins such asenzymes, growth factors, receptors, antibodies, and signaling moleculesdepend on their conformational structure for their biological activity.Other classes of proteins are primarily structural, e.g. collagen andcartilage, and do not possess biological activity per se.

Exposing biomolecules to various environments such as variations in pH,temperature, solvents, osmolality, etc., can irreversibly change ordenature the conformational state of the biomolecule, rendering itbiologically inactive. Some of the mechanisms involved in thedeactivation of these biomolecules include aggregation, oxidation,various types of bond cleavage including hydrolysis and deamidation, andvarious types of bond formation, including cross-linking and othercovalent binding, for example the rearrangement of disulfide bonds.

Bone morphogenetic proteins and the closely related growth anddifferentiation factors (in both monomeric and dimeric forms) belong tothe TGF-β superfamily of proteins. This class of proteins includesmembers of the family of bone morphogenetic proteins that were initiallyidentified by their ability to induce ectopic endochondral boneformation (see Cheng et al. “Osteogenic activity of the fourteen typesof human bone morphogenic proteins” J. Bone Joint Surg. Am. 85A: 1544-52(2003)). There are alternate names for several of these proteins, (seeLories et al., Cytokine Growth Factor Rev 16:287-98 (2005)). All membersof this family share common structural features, including a carboxyterminal active domain, and are approximately 97-106 amino acids inmature length. All members share a highly conserved pattern of cysteineresidues that create 3 intramolecular disulfide bonds and oneintermolecular disulfide bond. The active form can be either adisulfide-bonded homodimer of a single family member or a heterodimer oftwo different members. (see Massague Annu. Rev. Cell Biol. 6:957 (1990);Sampath, et al. J. Biol. Chem. 265:13198 (1990); Ozkaynak et al. EMBO J.9:2085-93 (1990); Wharton, et al. PNAS 88:9214-18 (1991); Celeste et al.PNAS 87:9843-47 (1990); Lyons et al. PNAS 86:4554-58 (1989), U.S. Pat.No. 5,011,691, and U.S. Pat. No. 5,266,683).

It is well established that many sugars stabilize biomolecules insolution and afford protection to isolated cells and biomolecules. Thesecompounds are well established as cryoprotectants and osmoregulators invarious species (see Yancey J. Exper. Biol. 208: 2819-30 (2005)). In thedevelopment of lyophilized pharmaceutical proteins, sugars (saccharidesand polyols) are often added to the formulation in order to improve thestability of the protein and prolong the shelf life. There are two maintheories on the mechanism of the stabilizing action of sugars: 1) thesugar excipients serve to dilute the proteins in the solid state,thereby decreasing protein-protein interactions and preventing moleculardegradation, such as aggregation, and 2) the sugar excipients provide aglassy matrix wherein protein mobility and hence reactivity areminimized. In both of these mechanisms, it is critical that the sugarremains in the amorphous, protein-contacting phase. Variousenvironmental factors, such as increased temperature and moisture, caninduce sugar crystallization. Thus, it is important to optimize theconditions and materials used to suit the particular biomolecule andsystem under consideration.

Lyophilization (freeze-drying) is a method commonly used to preservebiomolecules. Freeze-drying is generally thought to be more disruptiveto the biological activity of biomolecules than freeze-thawing ortemperature-induced denaturation. The magnitude of damage variesconsiderably with different biomolecules and different conditions, andvarious investigators have studied different systems. The freezing ofaqueous solutions creates an initial increase in solute concentrationsthat can be more damaging to labile compounds than the freezing itself.Excipients such as sugars, proteins, polymers, buffers, and surfactantscan be added to stabilize the activity of the biomolecule, but havelimited and varying degrees of success, depending on the system. Crowe,et al. describes the stabilization of dry phospholipid bilayers andproteins by sugars (Biochem. J. 242: 1-10 (1987)), and also reviews therecent understanding of the mechanisms of trehalose stabilization ofcells in “The trehalose myth revisited: Introduction to a symposium onstabilization of cells in the dry state” Cryobiology 43, 89-105 (2001).The current thinking is that there are two separate and differentrequirements for maintaining a viable and useful lyophilized protein: 1)the protein must be protected during the freezing process, and 2) theprotein must be protected during the subsequent drying andreconstitution. These are different requirements that are notnecessarily met by any one excipient or set of conditions.

Various researchers have reported on using various excipients to protectvarious biomolecules, for example Gloger, et al. (Intl. J. Pharm. 260:59-68 (2003)) described the lyoprotection of aviscumine using lowmolecular weight dextrans to stabilize the protein, and showed that thebuffer system and polysorbate 80 alone are suitable to protect theprotein during freezing, but dextran is needed to protect the proteinduring drying; Goodnough, et al. (Appl. Env. Biol. 58(10: 3426-28(1992)) investigated the stabilization of Botulinum toxin type A duringlyophilization using serum albumin as stabilizer and various otherexcipients, and reported that none of the excipients had any beneficialeffect, but by eliminating NaCl from the lyophilization mixture and bycontrolling the pH, the recovery of active toxin was dramaticallyimproved; Costantino, et al. (J. Pharm. Sci. 87(11): 1412-20 (1998))described the effects of various saccharides on the stability andstructure of lyophilized recombinant human growth hormone, and showedthat all of the excipients tested significantly improved the stabilityof the protein; Ramos et al. (Appl. Envir. Microbiol. 63(10): 4020-25(1997)) showed that 2-O-β-mannosylglycerate is effective in protectingseveral dehydrogenase enzymes isolated from various sources from thermalstress, and that the protection afforded by 2-O-β-mannosylglycerate wassimilar to or superior to trehalose for all of the enzymes studied, butwas not effective in protecting glutamate dehydrogenase isolated from P.furiosis; Brus, et al. (J. Control. Rel. 95:119-31 (2004)) investigatedthe stabilization of oligonucleotide-polyethylenimine (PEI) complexes byfreeze-drying, and reported that these complexes did not benefit fromthe addition of sugars such as sucrose or trehalose, but thatplasmid-PEI complexes did benefit from the addition of such sugars.These investigators report varying degrees of success, as measured byvarious methods on various biomolecules. None of these investigatorshave reported on the protection of BMP's.

Thus, there is conflicting evidence on what is an optimal combination ofexcipients to afford lyoprotection of biomolecules. There is not any onecombination of excipients that is optimal for all biomolecules, butrather a significant degree of experimentation is required to obtain thedesired results for the biomolecule under investigation. There remains aneed for a pharmaceutically acceptable combination of excipients toprotect BMP's during lyophilization, storage, and use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DSC profile of the trehalose formulation of rhGDF-5 asdescribed in example 6.

FIG. 2 shows the DSC profile of the mannitol formulation of rhGDF-5 asdescribed in example 7.

FIG. 3 shows the DSC profile of rhGDF-5 native protein.

FIG. 4 shows the polarized light microscopy of the trehalose formulationof rhGDF-5 as described in example 6.

FIG. 5 shows the polarized light microscopy of the mannitol formulationof rhGDF-5 as described in example 7.

FIG. 6 shows the rpHPLC profile of the rhGDF-5/trehalose/Glycineformulation after 6 months at 40° C./75% RH as described in example 12.

FIG. 7 shows the profile of the rpHPLC of the rhGDF-5/trehalose/HClformulation after 6 months at 40° C./75% RH as described in example 12.

FIGS. 8, 9, and 10 show the % protein recovery of the various bufferstested at storage at 5°, 25°, and 40° C. at various time points, asdescribed in example 12.

FIG. 11 shows the stability of Various Concentrations of rhGDF-5 atSelected Temperatures Lyophilized With 5% or 10% Trehalose in pH3Glycine Buffer, as described in example 14.

SUMMARY OF THE INVENTION

The present invention is generally directed to stabilizing BMP's invarious formulations and compositions, thereby preserving at least 60%of the biological activity and improving the storage conditionrequirements, for example temperature and humidity. The presentinvention comprises formulations primarily including trehalose as anexcipient for lyophilized compositions containing BMP and theirsubsequent storage and reconstitution, and further comprising otherexcipients including buffers and surfactants.

The present inventors have surprisingly discovered that trehalose issufficient and superior to other excipients to preserve the biologicalactivity of BMP's during and after lyophilization. In the stabilizationof many other biomolecules there is little difference among sugars as tothe amount of protection afforded, but for BMP's there is a greatdifference. This discovery provides for compositions to treat variousmusculoskeletal defects in a patient without the potential for adversereactions to additional excipients. The present inventors have alsosurprisingly discovered that the addition of antioxidants such asascorbic acid and glutathione do not increase the stability of the BMPlyophilized with trehalose, but rather detracts from the stabilityafforded by trehalose.

It is an object of the invention to utilize trehalose in an amount thatis sufficient to stabilize a lyophilized BMP, such that the BMP retainsat least 60% of the biological activity upon rehydration, with saidrehydrated liquid product being easily handled by the surgeon.

It is another object of the invention to utilize trehalose in an amountthat is sufficient to stabilize a lyophilized BMP, at least one BMP, andadditional excipients, said additional excipients selected from thegroup consisting of a buffer, a surfactant and mixtures thereof, suchthat the BMP retains at least 60% of the biological activity uponrehydration, with said rehydrated liquid product being easily handled bythe surgeon.

It is another object of the invention to utilize trehalose in an amountthat is sufficient to stabilize a lyophilized BMP, at least one BMP, andmorselized collagen fibers to provide compositions and methods ofpreparing a lyophilized biocompatible flowable material containing BMPthat is stable and retains at least 60% of the biological activity uponrehydration, such that the rehydrated product can be easily handled bythe surgeon.

It is another object of the invention to utilize trehalose in an amountthat is sufficient to stabilize a lyophilized BMP, at least one BMP, anda biocompatible matrix to provide compositions and methods of preparinga lyophilized biocompatible matrix containing BMP that is stable andretains at least 60% of the biological activity upon rehydration, suchthat the rehydrated product can be easily handled by the surgeon.Exemplary biocompatible matrices include collagen, mineralized collagen,salts of calcium phosphate, ceramics containing calcium, bone fromvarious sources including autogenic, allogenic, and xenogenic, andpolymers, including polylactide (PLA), polyglycolide (PGA), PLA-PGAco-polymers, polycarbonate, polycaprolactone and mixtures thereof.

It is another object of the invention to utilize trehalose in an amountthat is sufficient to stabilize a lyophilized BMP, at least one BMP, abiocompatible matrix, and additional excipients, said additionalexcipients selected from the group consisting of a buffer, a surfactantand mixtures thereof, to provide compositions and methods of preparing alyophilized biocompatible matrix containing BMP that is stable andretains at least 60% of the biological activity upon rehydration, suchthat the rehydrated malleable product can be easily handled by thesurgeon.

It is another object of the invention to utilize one or morelyoprotectants selected from the group consisting of trehalose, lowmolecular weight dextran, cyclodextrin, polyethylene glycol,polyethylene glycol ester and mixtures thereof, in an amount that issufficient to stabilize a lyophilized BMP, and at least one BMP toprovide compositions and methods of preparing a lyophilized BMP, suchthat the BMP retains at least 60% of the biological activity uponrehydration, with said rehydrated product being easily handled by thesurgeon.

It is another object of the invention to utilize one or morelyoprotectants selected from the group consisting of trehalose, lowmolecular weight dextran, cyclodextrin, polyethylene glycol,polyethylene glycol ester and mixtures thereof, at least one BMP, andcollagen to provide compositions and methods of preparing a lyophilizedbiocompatible collagen matrix containing BMP that is stable and retainsat least 60% of the biological activity upon rehydration, such that therehydrated malleable product can be easily handled by the surgeon.

It is another object of the invention to utilize one or morelyoprotectants selected from the group consisting of trehalose, lowmolecular weight dextran, cyclodextrin, polyethylene glycol,polyethylene glycol ester and mixtures thereof, at least one BMP, andmorselized collagen fibers to provide compositions and methods ofpreparing a lyophilized biocompatible flowable material containing BMPthat is stable and retains at least 60% of the biological activity uponrehydration, such that the rehydrated product can be easily handled bythe surgeon.

It is still another object of the invention to treat a patient utilizinga composition comprised of a lyophilized mixture of at least onelyoprotectant and at least one BMP. Such compositions are useful intreating a variety of musculoskeletal defects in order to enhance thehealing process, either by directly applying the reconstituted BMPsolution to a region of the anatomy of a patient, such as for example toa bone fracture, a bone gap, a bone void, an intervertebral disc, achondral defect, a tendon, a ligament, and the like, or applying thereconstituted BMP solution to a device to be implanted into the patient,for example a bone-contacting artificial implant such as an artificialhip, knee, shoulder, intervertebral disc, and the like, a tendon anchor,ligament anchor, suture, staple, and the like, a bone replacement cage,autologous bone chips, allogenic bone chips, xenogenic bone chips,demineralized bone chips, and the like.

Bulk forms of BMP in either aqueous solution or as a dry solid are notstable, and require cold storage below −20° C. to preserve thebiological activity of the protein. Since BMP is susceptible toaggregation, rearrangement of disulfide bonds, deamidation, andoxidation, a need is present for a formulation to preserve and protectthe biological activity of lyophilized BMP.

There is a need for a lyophilized BMP product with improved stabilityand storage.

There is a need for a lyophilized BMP product for reconstitution withaqueous solutions to be used for injection into soft tissue such as theintervertebral disc, non-articular and articular cartilage to promoteregeneration of such tissues.

There is a need for a lyophilized BMP product that is provided on animplantable biocompatible scaffold with the proper concentration of BMPfor the physician to use, thereby minimizing or eliminating many of therisks associated with handling, including contamination, improperdosage, and spillage, including waste and introduction to an undesiredsurgical site.

There is a need for a lyophilized BMP product that can be reconstitutedin a biocompatible flowable material that can be easily applied to asurgical site.

DETAILED DESCRIPTION OF THE INVENTION

Since the discovery of BMP, there has been considerable researchactivity to find a suitable composition for their therapeutic use intreating a variety of musculoskeletal defects and conditions. Currentlythere are products containing BMP that are sold as a lyophilized solid,which must be reconstituted to a liquid form and applied by thephysician to the scaffold to be implanted or to the surgical treatmentsite at the time of use. The current formulation of rhBMP-2 uses sucroseNF, glycine USP, L-glutamic acid FCC, sodium chloride USP, andpolysorbate 80 NF as excipients, and may be stored at room temperature(15-25° C.). The current formulation of OP-1 uses bovine collagen alone,and must be stored at 2-8° C. There are no published reports thatdescribe the efficacy of excipients on the stability of thereconstituted BMP.

Others have attempted to enhance the stability of BMP duringlyophilization by using mannitol, sucrose, and mixtures thereof, byembedding the BMP in polymer matrices such as PLGA, by addinganti-oxidants such as methionine, by adding other excipients such ashistidine, arginine, cyclodextrin, and bovine serum albumin, and byadding surfactants such as TWEEN 80, or combinations thereof. Theseattempts have met with varied degrees of limited success.

U.S. Pat. Nos. 5,318,898 and 5,516,654 disclose improved processes ofproducing BMP by using dextran sulfate in the culture medium, but do notdiscuss the mechanism of how the benefit is achieved or disclose anyother useful excipients to stabilize the proteins. In U.S. Pat. No.5,385,887 Yim et al. disclose lyophilized compositions and formulationsfor the delivery of BMP, with said compositions comprised of a BMP, asugar, glycine, and glutamic acid. Although Yim et al. disclose that thelyophilized formulations retain biological activity as evidenced by theW-20 Alkaline Phosphatase Assay, they do not disclose comparative dataon the formulations to show any quantitative benefits of any oneformulation over another. These inventors do not discuss or recognizethe superiority of trehalose over sucrose for lyoprotection of the BMP.

The present invention provides for compositions and methods of preparingand using stable formulations of BMP, useful for lyophilization,storage, and reconstitution with an aqueous solution to treat a patienttherewith. The present invention is described below relative toillustrative embodiments, and utilizes rhGDF-5 as the exemplary BMP.Those skilled in the art will appreciate that the present invention maybe implemented in a number of different applications and embodiments andis not specifically limited in its application to the particularembodiments depicted herein. The following examples illustrate some ofthe various embodiments and benefits of the present invention, howeverone skilled in the art will appreciate that other similar embodimentscan be made without deviating from the scope and intent of the presentinvention.

The present invention provides, in one aspect, a composition and methodfor preparing a stable lyophilized BMP for subsequent use in thesurgical treatment of bone and cartilage defects. As contemplatedherein, such a composition comprises at least one BMP and trehalose inan amount sufficient to stabilize the BMP. Such compositions are usefulin treating a variety of musculoskeletal defects by directly applyingthe reconstituted protein solution either directly to a region of theanatomy of a patient, such as for example to a bone fracture, a bonegap, a bone void, an intervertebral disc, an intervertebral disc spaceas surgically prepared for fusion, a chondral defect, a tendon, aligament, and the like, or to a material to be implanted into thepatient in contact with bone or cartilage, such as an artificial hip, anartificial knee, an artificial shoulder, an artificial intervertebraldisc, a tendon anchor, a ligament anchor, a suture, a staple, a bonecage, autologous bone chips, allogenic bone chips, xenogenic bone chips,demineralized bone chips, and the like.

As used herein, the terms “morphogen”, “bone morphogen”, “bonemorphogenic protein”, “bone morphogenetic protein”, “BMP”, “osteogenicprotein”, “osteogenic factor”, “Growth & Differentiation Factor”, and“GDF” embrace the class of proteins typified by rhGDF-5. It will beappreciated by one having ordinary skill in the art, however, thatrhGDF-5 is merely representative of the TGF-β family subclass of truetissue morphogens capable of acting as BMP, and is not intended to limitthe description. The term “cryoprotectant” is used to refer to amolecule capable of stabilizing a biomolecule during freezing, and isequivalent in the current context with the term “lyoprotectant”, whichrefers to a molecule capable of stabilizing a biomolecule duringfreeze-drying (lyophilization). As used herein, the term “morselized”refers to the product obtained by, and “morselization” to the process ofcutting, chopping, severing, grinding, pulverizing, or otherwisereducing the size of an amount of a biocompatible matrix, for examplecollagen, such that the overall size of the individual particles orfibers are reduced. As used herein, the term “excipient” refers to atleast one additional compound added to at least one BMP, with saidadditional compound selected from the group consisting of amino acids,proteins, buffers, surfactants and mixtures thereof. The terms “ceramic”and “ceramics containing calcium” are understood to mean synthetic bonesubstitute materials known in the art, and include for example Bioglass™and various compositions containing primarily silica, alumina, andmixtures thereof, with smaller amounts of calcium, barium, strontium,magnesium, carbonate, sodium, potassium, fluoride, and other ions usedto modify the properties of the synthetic bone material. The term “saltsof calcium phosphate” is understood to mean various compositions ofcalcium phosphate useful for bone substitute materials, including, butnot limited to hydroxyapatite, tricalcium phosphate, brushite, monetite,and various other stoichiometric ratios of calcium and phosphate usefulfor bone substitute materials, including calcium phosphate compositionswith the addition of smaller amounts of other ions, such as magnesium,barium, strontium, carbonate, sodium, potassium, fluoride, etc. tomodify the properties, as is commonly known in the art.

It has been known that rhGDF-5 has poor solubility at neutral pH in therange of pH 4.5 to pH 10.5. It would be difficult to formulate andmanufacture rhGDF-5 products in this pH range. Therefore the inventorsdesigned a study to evaluate the solubility of rhGDF-5 in pH 3 and pH 4buffers, which is critical to select a suitable pH range for thedevelopment of protein formulations. The study results are described inexample 11. The solubility of rhGDF-5 depends not only on the pH of thebuffer, but also depends on the ionic strength of the buffer solution.At pH 4, the rhGDF-5 solutions at approximately 10 mg/mL were hazy in 5and 10 mM sodium phosphate buffers, while in 50 and 100 mM sodiumphosphate buffers the rhGDF-5 formed large particles and finallyprecipitated out. In another study (data not shown) when rhGDF-5 wasformulated at 3.5 mg/mL at pH 3.5 and pH 4 of 5 mM phosphate buffer, thesolutions were also hazy.

The solubility of a protein substance is usually determined by measuringthe protein concentration after centrifugation or filtration of an oversaturated/precipitated solution. However, some hazy protein solutionsare difficult to centrifuge or filter. Even after a hazy solution issubjected to centrifugation or filtration (0.22 μm) to remove theinsoluble particles, quite often it is unsuccessful as the filtratestill looks hazy because the particles are so fine and some times theprotein sticks to filter surface, thus the filtrate loses most of theprotein. Therefore, it would be difficult to get a clear solution whenrhGDF-5 is formulated at 3.5 mg/mL or 10 mg/mL in pH 3.5 or pH 4buffers.

When rhGDF-5 was formulated at 10 mg/ml in 5 mM, 10 mM and 25 mM sodiumphosphate solutions at pH 3.0, the protein solution was clear; butrhGDF-5 at 10 mg/ml in higher ionic strength solutions such as 50 mM and100 mM sodium phosphate, the rhGDF-5 solutions were hazy. Thus, in apreferred embodiment the rhGDF-5 should be formulated in a low ionicstrength buffer at approximately pH 3.0.

In one embodiment according to the present invention the composition canbe prepared by lyophilizing an aqueous mixture of at least one BMPtogether with an amount of trehalose sufficient to stabilize the BMP,with the dry weight ratio of trehalose to BMP being in the range ofabout 1 mg to about 500 mg trehalose per 1 mg BMP, and more preferablyin the range of about 5 mg to about 200 mg trehalose per 1 mg BMP forbiocompatible matrix containing products. The addition of trehaloseprovides for improved solubility and stability of the protein inlyophilized formulations. Lyophilization is performed according to thepractice as generally known in the art.

In another embodiment the composition according to the present inventioncan be prepared by lyophilizing an aqueous mixture of at least one BMP,an amount of trehalose sufficient to stabilize the BMP, and a bufferingagent. The addition of a buffering agent provides for improvedsolubility and stability of the protein in lyophilized formulations.Biocompatible buffering agents known in the art include glycine; sodium,potassium, or calcium salts of acetate; sodium, potassium, or calciumsalts of citrate; sodium, potassium, or calcium salts of lactate; sodiumor potassium salts of phosphate, including mono-basic phosphate,di-basic phosphate, tri-basic phosphate and mixtures thereof. Thebuffering agents could additionally have glycine added to thecomposition to function as a bulking agent. The glycine would be addedin a ratio of about 0.04 mg to about 200 mg glycine per 1 mg BMP, andmore preferably from about 1 mg to about 80 mg glycine per 1 mg BMP. Theaddition of buffering and bulking agents provides for slightly superiorstability of the protein over compositions having trehalose alone, withthe pH being controlled within about 2.0 to about 5.0 pH units, and morepreferably within about 2.5 to about 3.5 pH units.

In an alternate embodiment the composition and method according to thepresent invention can be prepared by lyophilizing an aqueous mixture ofat least one BMP, an amount of trehalose sufficient to stabilize theBMP, a buffering agent, and a surfactant selected from the groupconsisting of polysorbate 80, polysorbate 20 and mixtures thereof. Thesurfactant would be added in a concentration of from about 0.001 mg toabout 0.2 mg per 1 mg of BMP. The addition of surfactant providesadditional stabilization to the protein by altering the solubility andlyophilization characteristics. Lyophilization would be performedaccording to the practice as generally known in the art.

In another embodiment of the present invention, a composition and methodfor preparing a stable lyophilized BMP is comprised of at least one BMP,the lyoprotectant trehalose in an amount sufficient to stabilize the atleast one BMP, and at least one additional excipient, said additionalexcipient selected from the group consisting of buffers and surfactants.The addition of such buffers and surfactants provides for an incrementalimprovement in the stability of the lyophilized BMP over compositionshaving trehalose as the sole excipient.

In an alternate embodiment, the composition and method according to thepresent invention can be prepared by depositing a solution of at leastone BMP and at least one excipient onto lyophilized collagen prior tolyophilization of the BMP/collagen mixture. The collagen can optionallybe either cross-linked or mineralized, or both cross-linked andmineralized, such as is provided by the material known as Healos® anddescribed in U.S. Pat. Nos. 5,972,385; 5,866,165; 5,776,193; 5,455,231;and 5,231,169. The compositions provided in this embodiment areparticularly useful in treating medical conditions in the field oforthopedics and provide a pliable, malleable material that the physiciancan easily place into a surgical site to generate bone, cartilage, ortendon. The BMP/collagen mixture can be reconstituted with aqueoussolutions, including sterile water, saline solution, and bone marrowaspirate, and directly applied to defect sites in a patient, such asbone fractures, bone gaps, bone voids, the intervertebral disc spacesurgically prepared for spinal fusion. Additionally, the BMP/collagenmixture can be used for filling the space in between bone chips andimplants placed into the intervertebral disc space during spinal fusionsurgery, areas with damaged or missing cartilage, such as torn ordamaged tendons, torn or damaged ligaments, chondral defects inarticulating cartilage, and sub-chondral defects in articulatingcartilage.

In an alternate embodiment, the composition and method according to thepresent invention can be utilized by preparing a lyophilized mixture ofat least one BMP and at least one excipient, reconstituting thelyophilized BMP mixture with water, saline solution, or bone marrowaspirate, and placing the reconstituted BMP solution onto lyophilizedcollagen prior to surgical implantation of the BMP/collagen mixture. Thecollagen can optionally be either cross-linked or mineralized, or bothcross-linked and mineralized, such as is provided by the material knownas Healos®. The compositions and methods provided in this embodiment areparticularly useful in treating medical conditions in the field oforthopedics and provide a pliable, malleable material that the physiciancan easily place into a surgical site to generate bone, cartilage, ortendon. The BMP/collagen mixture can be directly applied to defect sitesin a patient, such as bone fractures, bone gaps, bone voids, theintervertebral disc space surgically prepared for spinal fusion, fillingthe space in between bone chips and implants placed into theintervertebral disc space during spinal fusion surgery, areas withdamaged or missing cartilage, such as torn or damaged tendons, torn ordamaged ligaments, chondral defects in articulating cartilage, andsub-chondral defects in articulating cartilage. The compositions andmethods provided in this embodiment are also particularly useful forease of storage and preparation by virtue of having the BMP as aseparate component and container from the collagen material.

In an alternate embodiment the composition and method according to thepresent invention can be prepared by depositing a solution of at leastone BMP and at least one excipient onto lyophilized morselized collagenprior to lyophilization of the BMP/morselized collagen mixture. Themorselized collagen could optionally be either cross-linked ormineralized, or both cross-linked and mineralized. Such morselizationprovides for small collagen fibers of about 25 microns in diameter byabout 110 microns length, which yields a flowable composition suitablefor injection into a surgical site. Reconstitution of such a compositioncan be performed using a mixture of an aqueous solution such as sterilewater, saline, or bone marrow aspirate, and collagen gel, with thecollagen gel providing control of the viscosity of the reconstitutedproduct. The collagen gel contains from about 0.1% to about 30 w/wcollagen, and more preferably from about 0.3% to about 3.0% w/wcollagen, with the viscosity of the collagen gel preferably from about10 cP to about 400 cP, and more preferably from about 70 cP to about 100cP. The pH of the collagen gel is preferably from about 4.0 pH units toabout 8.0 pH units. Such a composition is useful for treating a varietyof musculoskeletal conditions, including but not limited to bonefractures, bone gaps, bone voids, the intervertebral disc spacesurgically prepared for spinal fusion, filling the space in between bonechips and implants placed into the intervertebral disc space duringspinal fusion surgery, areas with damaged or missing cartilage, such astorn or damaged tendons, torn or damaged ligaments, chondral defects inarticulating cartilage, and sub-chondral defects in articulatingcartilage.

In an alternate embodiment the composition and method according to thepresent invention can be utilized by preparing a lyophilized mixture ofat least one BMP and at least one excipient, reconstituting thelyophilized BMP mixture with water or saline solution, and injecting thereconstituted BMP solution into the intervertebral disc. Thecompositions and methods provided in this embodiment are particularlyuseful in treating the intervertebral disc.

EXAMPLES OF THE INVENTION

In the following examples, the experimental methods used were asfollows:

For RP-HPLC purity studies, reconstituted rhGDF-5 test samples werediluted to a concentration of 0.1 mg/ml with 10 mM HCl and subjected toreversed phase-HPLC on a Vydac 218TP52 column at 50° C. and a flow rateof 0.3 ml/min. rhGDF-5 is eluted using a gradient of acetonitrile in0.15% trifluoroacetic acid using UV detection at 214 nm.

For Circular dichroism (CD) studies, Circular Dichroism was performed onan AVIV Model 60DS Circular Dichroism Spectropolarimeter. Baselineplacebo runs with corresponding excipient scans were subtracted from thesample scans. The scans were normalized using Mean Residue Weight (valueof 115) and inserting it into the equation:[⊖]=[0.1×M _(residue)]/[conc. (mg/ml)×light path]

The value of [⊖] was calculated at each wavelength to give mean residueellipticities. Finally, an estimate of secondary structure wasdetermined using the program PROSEC v.2.1 (copyrighted in 1987 by AVIVAssociates).

Differential scanning calorimetry (DSC) was performed on a MicroCalVP-DSC instrument. The scan rate was 60° C./h. The temperature range was5-100° C. Instrument baseline scan (placebo data) was subtracted fromtest sample heat scan. The protein concentration was 0.33 mg/ml.

Polarized Light Microscopy (PLM) was used for Crystallinity Assessment.A trace amount of the solid sample was taken out of the vial in a dryair bag with a relative humidity of 1%. The solid sample was spread on aglass slide and one drop of silica oil was dropped onto the solidsample. Then the slide was investigated with a Zeiss Optical Microscopeequipped with a Sony CCD-IRIS/RGB Color Video Camera and polarized lightaccessory. Flash Bus FBG software was used to capture images.

Bulk rhGDF-5 was received from Biopharm in a frozen format at −80° C. ata concentration of 3.8 mg/ml in 10 mM HCl. The frozen bulk protein wasthawed over night at 2-8° C. before using in formulations.

Example 1

Healos® strips (non-sterile) with rhGDF-5 (0.5 mg/ml, 5 ml/strip) andtrehalose 50 mg/ml. Each strip had 2.5 mg of rhGDF-5 and 250 mg oftrehalose.

Preparation of Trehalose Solution:

25.48 g of trehalose dihydrate was carefully weighed and transferredinto a sterile polypropylene bottle, to which 350 ml of purified waterwas added at room temperature and stirred slowly until a clear solutionwas obtained. To the clear solution, 0.1 N HCl was added drop by drop toadjust the pH to 3.9, then the volume was adjusted with purified waterto obtain 400 ml final volume. The pH was measured and found to be 4.2.The solution was filtered through a 0.22-micron filter and was useddirectly to dilute the protein solution.

Dilution of rhGDF-5 Solution with Trehalose Solution:

22.39 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which trehalose solution was added carefully toadjust the volume to 150 ml; the pH was measured and found to be 2.5.The solution was stirred for 15 minutes at room temperature. The UVextinction coefficient was obtained to accurately calculate the proteinconcentration. Based on the UV reading, more trehalose solution wasadded to obtain the desired concentration of 0.5 mg/ml in 170 mlsolution; the pH was measured and found to be 2.7; the UV readingindicated 0.499 mg/ml protein content.

The rhGDF-5/trehalose solution was filtered through a 0.22-micron filterand was used directly to dispense onto Healos® strips. Using sterilepipettes, 2.5 ml of rhGDF-5/trehalose solution was dispensed onto stripsequally at 2 spots for a total of 5 ml of rhGDF-5/trehalose solution pereach strip. The strips were inserted into small 2 cm by 5 cm PETG trays,and the small trays were inserted into large PETG trays and lyophilized.Each large tray accommodates 24 strips.

TABLE 1a Stability of Healos ® with trehalose (250 mg) plus rhGDF-5 (2.5mg) per strip at 25° C. (Example 1) Pa- ram- 0 0 3 6 9 12 Test etermonths months months months months months RP- % 89.54 82.26 81.49 77.9876.57 72.19 HPLC main peak RP- % 0.00 3.01 5.08 4.53 5.40 6.60 HPLC ag-gre- gates

TABLE 1b Stability of Healos ® with trehalose (250 mg) plus rhGDF-5 (2.5mg) per strip at 2-8° C. (Example 1) Pa- ram- 0 0 3 6 9 12 Test etermonths months months months months months RP- % 89.54 88.22 90.84 85.4588.70 87.61 HPLC main peak RP- % 0.00 0.00 0.00 0.00 0.00 0.00 HPLC ag-gre- gates

Example 2

Healos® strips (non-sterile) with rhGDF-5 (0.5 mg/ml, 5 ml/strip) andmannitol 50 mg/ml. Each strip had 2.5 mg of rhGDF-5 and 250 mg ofmannitol.

Preparation of Mannitol Solution:

23.03 g of mannitol was carefully weighed and transferred into a sterilepolypropylene bottle, to which 350 ml of purified water was added atroom temperature and stirred slowly until a clear solution was obtained.The pH was measured and found to be 7.2; 0.1 N HCl was added drop bydrop to adjust the pH to 3.8; then the volume was adjusted with purifiedwater to obtain 400 ml final volume. The pH was measured and found to be3.9. The solution was filtered through a 0.22-micron filter and was useddirectly to dilute the protein solution.

Dilution of rhGDF-5 Solution with Mannitol Solution:

22.37 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which mannitol solution was carefully added toadjust the volume to 150 ml. The pH was measured and found to be 2.7.The solution was stirred for 15 minutes at room temperature. The UVextinction coefficient was obtained to calculate an accurate proteinconcentration. Based on the UV reading, more mannitol solution was addedto obtain the desired concentration of 0.5 mg/ml in 170 ml of solution;the pH was measured and found to be 2.8; the UV reading indicated 0.493mg/ml protein content.

The rhGDF-5/mannitol solution was filtered through a 0.22-micron filterand was used directly to dispense onto Healos® strips. Using sterilepipettes, 2.5 ml of rhGDF-5/mannitol solution was dispensed onto stripsequally at 2 spots for a total of 5 ml of rhGDF-5/mannitol solution pereach strip. The strips were inserted into small 2 cm by 5 cm PETG trays,and the small trays were inserted in large PETG trays and lyophilized.Each large tray accommodates 24 strips.

TABLE 2a Stability of Healos ® with mannitol (250 mg) plus rhGDF-5 (2.5mg) per strip at 25° C. (Example 2) Pa- ram- 0 1 3 6 9 12 Test etermonths month months months months months RP- % 89.54 78.89 63.10 51.48At six months, HPLC main the main peak peak was markedly RP- % 0.00 5.6712.24 12.56 decreased and HPLC ag- accumulation of gre- aggregates wasgate increased. The stability studies were terminated at six months.

TABLE 2b Stability of Healos ® with mannitol (250 mg) plus rhGDF-5 (2.5mg) per strip at 2-8° C. (Example 2) Pa- ram- 0 1 3 6 9 12 Test etermonths month months months months months RP- % 89.71 89.12 86.26 81.0282.97 79.78 HPLC main peak RP- % 0.00 0.00 2.70 3.21 4.01 4.12 HPLC ag-gre- gates

Example 3

Healos® strips (sterile) with rhGDF-5 (0.5 mg/ml, 5 ml/strip) andtrehalose 100 mg/ml. Each strip had 2.5 mg of rhGDF-5 and 500 mg oftrehalose.

Preparation of Trehalose Solution:

25.49 g of trehalose dihydrate was carefully weighed and transferredinto a sterile polypropylene bottle, to which 190 ml of purified waterwas added at room temperature and stirred slowly until a clear solutionwas obtained. The clear trehalose solution pH was measured and found tobe 6.2. HCl was not added to the trehalose solution to adjust the pH.The volume was adjusted with purified water to obtain 200 ml finalvolume. The pH was measured and found to be 6.3. The solution was useddirectly to dilute the protein solution.

Dilution of rhGDF-5 Solution with Trehalose Solution:

23.03 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which trehalose solution was added carefully toadjust the volume to 170 ml. The pH was measured and found to be 3.0.The solution was stirred for 15 minutes at room temperature. The UVextinction coefficient was obtained to accurately calculate the proteinconcentration. Based on the UV reading, more trehalose solution wasadded to obtain the desired concentration of 0.5 mg/ml in 175 ml ofsolution; the pH was measured and found to be 3.0; the UV readingindicated 0.518 mg/ml protein concentration.

The rhGDF-5/trehalose solution was filtered through a 0.22-micron filterand was used directly to dispense onto sterile Healos® strips. Usingsterile pipettes, 2.5 ml of rhGDF-5/trehalose solution was dispensedonto strips equally at 2 spots for a total of 5 ml of rhGDF-5/trehalosesolution per each strip. The strips were placed on steel trays, whichwere carefully packed into sterile double pouches and transferred forlyophilization under sterile conditions.

TABLE 3a Stability of Healos ® with trehalose (500 mg)/rhGDF-5 (2.5 mg)per strip at 2-8° C. (Example 3) 0 1 3 6 Test Parameter months monthmonths months RP- % main peak 88.5 83.9 90.0 78.9 HPLC RP- % aggregates0.0 0.0 0.0 0.0 HPLC

Example 4

Healos® strips (sterile) with low dose rhGDF-5 (5 ml/strip, 0.5 mg/ml),trehalose 40 mg/ml and glycine 10 mg/ml. Each strip had 2.5 mg ofrhGDF-5, 200 mg of trehalose and 50 mg of glycine.

Preparation of Trehalose/Glycine Solution:

17.84 g of trehalose dihydrate and 4.03 g of glycine were carefullyweighed and transferred into a sterile polypropylene bottle, to which300 ml of purified water was added at room temperature and stirredslowly until a clear solution was obtained. The pH was measured andfound to be 5.5. Without adding any acid, the volume was adjusted to 350ml with purified water. The pH was measured and found to be 5.5.

Dilution of rhGDF-5 Solution with Trehalose/Glycine Solution:

39.47 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which trehalose/glycine solution was addedcarefully to adjust the volume to 295 ml. The pH was measured and foundto be 4.1. The solution was stirred for 15 minutes at room temperature.The UV extinction coefficient was obtained to accurately calculate theprotein concentration. Based on the UV reading, more trehalose solutionwas added to obtain the desired concentration of 0.5 mg/ml in 300 ml ofsolution; the pH was measured and found to be 4.1; the UV readingindicated 0.507 mg/ml protein concentration.

The solution was filtered through a 0.22-micron filter, and the solutionwas used directly to dispense on sterile Healos® strips. Using sterilepipettes, 2.5 ml of rhGDF-5/trehalose/glycine solution was dispensedonto strips equally at 2 spots for a total of 5 ml of rhGDF-5 solutionper each strip. The strips were placed on steel trays, which werecarefully packed into sterile double pouches and transferred forlyophilization under sterile conditions.

TABLE 4a Stability of Healos ® with trehalose (200 mg)/rhGDF-5 (2.5mg)/Glycine (50 mg) per strip at 2-8° C. (Example 4) 0 1 3 6 TestParameter months month months months RP- % main peak 87.9 83.5 86.0 80.1HPLC RP- % aggregates 0.0 0.0 0.0 0.0 HPLC

TABLE 4b Stability of Healos ® with trehalose (200 mg)/rhGDF-5 (2.5mg)/Glycine (50 mg) per strip at 25° C. (Example 4) 0 1 3 6 TestParameter months month months months RP- % main peak 87.9 78.7 78.1 67.7HPLC RP- % aggregates 0.00 0.00 0.00 0.00 HPLC

Example 5

Healos® strips (sterile) with rhGDF-5 (0.5 mg/ml, 2.5 mg/strip),trehalose 40 mg/ml, glycine 10 mg/ml and polysorbate 0.1 mg/ml. Eachstrip had 2.5 mg of rhGDF-5, 200 mg of trehalose, 50 mg of glycine and0.5 mg of polysorbate 80.

Preparation of Polysorbate 80 Solution:

23.03 mg of polysorbate 80 was weighed into a 50 ml sterile disposabletube, to which 25 ml of purified water was added and vortexed for 2minutes to obtain a homogenous solution.

Preparation of Trehalose/Glycine/Polysorbate Solution:

10.19 g of trehalose dihydrate and 2.303 g of glycine were carefullyweighed and transferred into a sterile polypropylene bottle, to whichthe 25 ml polysorbate 80 solution from above was added. The polysorbatetube was rinsed 2 times with 25 ml of purified water and the rinsestransferred to the trehalose/glycine/polysorbate solution. An additionalamount of purified water was added to the trehalose/glycine/polysorbatesolution for a total volume of 190 ml. The solution was stirred for 2minutes to obtain a clear solution. The pH of the solution was measuredand found to be 5.6; the volume was adjusted to 200 ml with purifiedwater. The pH was measured and found to be 5.5.

Dilution of rhGDF-5 Solution with Trehalose/Glycine/PolysorbateSolution:

23.03 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which the trehalose/glycine/polysorbate solutionwas added carefully to adjust the volume to 170 ml. The pH was measuredand found to be 4.1. The solution was stirred for 15 minutes at roomtemperature. The UV extinction coefficient was obtained to accuratelycalculate the protein concentration. Based on the UV reading, moretrehalose/glycine/polysorbate solution was added to obtain the desiredconcentration of 0.5 mg/ml in 175 ml of solution; the pH was measuredand found to be 4.1; the UV reading indicated 0.510 mg/ml proteinconcentration.

The solution was filtered through a 0.22-micron filter was used directlyto dispense onto sterile Healos® strips in a laminar flow hood underaseptic conditions. Using sterile pipettes, 2.5 ml ofrhGDF-5/trehalose/glycine/polysorbate solution was dispensed onto stripsequally at 2 spots for a total of 5 ml ofrhGDF-5/trehalose/glycine/polysorbate solution per each strip. Thestrips were placed on steel trays, which were carefully packed intosterile double pouches and transferred for lyophilization under sterileconditions.

TABLE 5a Stability of Healos ® with trehalose (200 mg)/rhGDF-5 (2.5mg)/Glycine (50 mg)/Polysorbate 80 (0.5 mg) per strip at 2-8° C.(Example 4) 0 1 3 6 Test Parameter months month months months RP- % mainpeak 88.4 84.3 86.8 82.2 HPLC RP- % aggregates 0.0 0.0 0.0 0.0 HPLC

Example 6

Lyophilized vial product of rhGDF-5 (0.5 mg/ml) plus trehalose (50mg/ml)

Preparation of Trehalose Solution:

A sterile polypropylene bottle was charged with 12.16 g of trehalosedihydrate and magnetic stir bar, to which 190 ml of purified water wasadded at room temperature. The solution was stirred at room temperatureuntil the trehalose was completely dissolved. The pH was measured andfound to be 6.5. To the clear trehalose solution, 0.1 N HCl was addeddrop by drop to adjust the pH to 5.8. The volume was adjusted to 200 mlwith purified water; the pH was measured and found to be 5.5. Thesolution was filtered through 0.22-micron filter and was used directlyto dilute the protein solution.

Dilution of rhGDF-5 Solution with Trehalose Solution:

14.47 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which trehalose solution was slowly added to afinal volume of 100 ml while swirling the bottle. The solution wasswirled occasionally at room temperature for 15 minutes; the pH wasmeasured and found to be 3.0. Based on the UV reading, more trehalosesolution was added to obtain the desired concentration of 0.5 mg/ml in110 ml of solution; the pH was measured and found to be 3.1; the UVreading indicated 0.510 mg/ml protein concentration. The solution wasfiltered through a 0.22-micron filter and was used directly to dispenseinto vials.

Filling vials: 1.1 ml of rhGDF-5/trehalose solution was dispensedmanually into 5 ml Type 1 flint glass vials, and each vial was partlyclosed with a stopper prior to loading into the lyophilizer. Afterlyophilization, the stoppers were pressed and crimped. The product wasobtained as white to off-white cake.

TABLE 6a Stability of vial of rhGDF-5 (0.5 mg/ml) plus trehalose (50mg/ml) at 2-8° C. (Example 6) 0 1 3 6 9 12 Test Parameter months monthmonths months months months Cake white to off- white white white whitewhite white Appearance & white; intact to off- to off- to off- to off-to off- to off- Integrity white; white; white; white; white; white;intact intact intact intact intact intact Reconstitution <2 min 1.130.28 1.32 0.38 0.44 0.34 Time, minutes pH of 2.0 to 3.5 3.0 2.5 2.8 2.82.9 2.9 Reconstituted Solution Protein mg/ml 0.49 0.46 0.46 0.48 0.470.45 Concentration RP-HPLC % main 90.74 88.58 92.91 92.96 92.77 93.19peak RP-HPLC % 0.00 0.00 0.00 0.00 0.00 0.00 aggregates

TABLE 6b Stability of vial of rhGDF-5 (0.5 mg/ml) plus trehalose (50mg/ml) at 25° C. (Example 6) 0 1 3 6 9 12 Test Parameter months monthmonths months months months Cake white to off- white white white whitewhite white Appearance & white; intact to off- to off- to off- to off-to off- to off- Integrity white; white; white; white; white; white;intact intact intact intact intact intact Reconstitution <2 min 1.130.26 1.44 0.38 0.52 0.33 Time, minutes pH of 2.0 to 3.5 3.0 2.5 2.8 2.92.8 2.9 Reconstituted solution Protein mg/ml 0.49 0.46 0.46 0.47 0.450.45 Concentration RP-HPLC % main 90.74 86.24 87.75 85.39 84.19 82.45 %main peak peak RP-HPLC % 0.00 0.00 0.00 0.00 0.00 0.00 % aggregatesaggregates

Example 7

Lyophilized vial product of rhGDF-5 (0.5 mg/ml) plus mannitol (50 mg/ml)

Preparation of Mannitol Solution:

A sterile polypropylene bottle was charged with 11.52 g of mannitol anda magnetic stir bar, to which 185 ml of purified water was added at roomtemperature. The mixture was stirred for 10 minutes at room temperatureuntil the mannitol was completely dissolved. The pH was measured andfound to be 6.6. To the clear solution, 0.1 N HCl was added drop by dropto adjust the pH to 5.5. The volume was adjusted to 200 ml with purifiedwater; the pH was measured and found to be 5.7. The solution wasfiltered through a 0.22-micron filter and was used directly to dilutethe protein solution.

Dilution of rhGDF-5 Solution with Mannitol Solution:

To a polypropylene flask, 14.48 ml of rhGDF-5 solution was carefullytransferred; to which the mannitol solution was added carefully to avolume of 100 ml. The solution was stirred for 15 minutes at roomtemperature. The UV extinction coefficient was obtained to accuratelycalculate the protein concentration. Based on the UV reading, moremannitol solution was added to obtain the desired protein concentrationof 0.5 mg/ml in 110 ml of solution; the pH was measured and found to be3.1; the UV reading indicated 0.498 mg/ml protein concentration. Thesolution was filtered through a 0.22-micron filter and was used directlyto dispense into vials.

Filling vials: 1.1 ml of mannitol/rhGDF-5 solution was dispensedmanually into 5 ml Type 1 flint glass vials, and each vial was partlyclosed with a stopper prior to loading into the lyophilizer. Afterlyophilization, the stoppers were pressed and crimped. The product wasobtained as white to off-white cake.

TABLE 7a Stability of vial of rhGDF-5 (0.5 mg/ml) plus mannitol (50mg/ml) at 2-8° C. (Example 7) 0 1 3 6 9 12 Test Parameter months monthmonths months months months Cake white to off- white white white whiteThe stability data Appearance & white; intact to off- to off- to off- tooff- at 6 months Integrity white; white; white; white; were not intactintact intact intact promising as Reconstitution <2 min 0.95 0.26 0.390.22 evidenced by the Time, minutes decrease in the pH of 2.0 to 4.0 3.53.2 3.3 3.9 main peak and Reconstituted the increase in Solutionaggregates, Protein mg/ml 0.41 0.39 0.37 0.36 hence the Concentrationstability studies RP-HPLC % main 89.85 86.65 82.04 53.59 were terminatedpeak at 6 months. RP-HPLC % 0.00 0.00 4.3 7.82 aggregates

TABLE 7b Stability of vial of rhGDF-5 (0.5 mg/ml) plus mannitol (50mg/ml) at 25° C. (Example 7) 0 1 3 6 9 12 Test Parameter months monthmonths months months months Cake white to off- white white white whiteThe stability data Appearance & white; intact to off- to off- to off- tooff- at 6 months Integrity white; white; white; white; were not intactintact intact intact promising, Reconstitution <2 min 0.95 0.35 0.350.28 hence the time, minutes stability studies pH of 2.0 to 4.0 3.5 3.43.6 4.0 were terminated Reconstituted at 6 months. solution Proteinmg/ml 0.41 0.32 0.28 0.26 Concentration RP-HPLC % main 89.85 34.31 26.0434.62 peak RP-HPLC % 0.00 7.05 14.21 17.30 aggregates

Example 8

Lyophilized vial product of rhGDF-5 (0.5 mg/ml) plus trehalose (50mg/ml) in glycine-HCl pH 3.0 buffer.

Preparation of Trehalose Solution:

A sterile polypropylene bottle was charged with 12.16 g of trehalosedihydrate and a magnetic stir bar, to which 200 ml of 5 mM glycine-HClbuffer pH 3.0 was added at room temperature. The solution was stirred atroom temperature until the trehalose was completely dissolved. The pH oftrehalose/glycine solution was 3.1. The solution was filtered through0.22-micron filter and was used directly to dilute the protein solution.

Dilution of rhGDF-5 Solution with Trehalose Solution:

Bulk rhGDF-5 solution was dialyzed against a 5 mM glycine-HCl bufferover night using a 3000 M.W. cut-off membrane at 2-8° C. After dialysisthe solution was slightly concentrated to 3.8 mg/ml. 14.47 ml of rhGDF-5solution was carefully transferred to a polypropylene flask, to whichtrehalose-glycine solution was slowly added to a final volume of 100 mlwhile swirling the bottle. The solution was swirled occasionally at roomtemperature for 15 minutes; the pH was measured and found to be 3.0.Based on the UV reading, more trehalose-glycine solution was added toobtain the desired protein concentration of 0.5 mg/ml in 110 ml ofsolution; the pH was measured and found to be 3.0; the UV readingindicated 0.507 mg/ml protein concentration. The solution was filteredthrough a 0.22-micron filter and was used directly to dispense intovials.

Filling vials: 1.1 ml of rhGDF-5/trehalose solution was dispensedmanually into 5 ml Type 1 flint glass vials, and each vial was partlyclosed with a stopper prior to loading into the lyophilizer. Afterlyophilization, the stoppers were pressed and crimped. The product wasobtained as white to off-white cake.

TABLE 8 Stability of vial of rhGDF-5 (0.5 mg/ml) plus trehalose (50mg/ml) in glycine-HCl buffer pH 3.0 (Example 8) Solution Proteinappearance main peak % Time and Cake after % recovery aggregatesTemperature appearance reconstitution HPLC HPLC Time = zero Solid,white- clear 100 0 to off-white, 1 month, Solid, white clear 100 0 50 C.to off-white 2 month, Solid, white clear 99.6 0 50 C. to off-white 3month, Solid, white clear 100 0 50 C. to off-white 1 month, Solid, whiteclear 100 0 250 C. to off-white 2 month, Solid, white clear 99.0 0 250C. to off-white 3 month, Solid, white clear 99.3 0 250 C. to off-white 1month, Solid, white clear 98.7 0 400 C. to off-white 2 month, Solid,white clear 99.1 0 400 C. to off-white 3 month, Solid, white clear 98.90 400 C. to off-white

Example 9

Lyophilized vial product of rhGDF-5 (0.5 mg/ml) plus trehalose (50mg/ml) in phosphate buffer at pH 3.0.

Preparation of Trehalose Solution:

A sterile polypropylene bottle was charged with 12.16 g of trehalosedihydrate and a magnetic stir bar, to which 200 ml of 5 mM phosphatebuffer pH 3.0 was added at room temperature. The solution was stirred atroom temperature until the trehalose was completely dissolved. The pH ofthe trehalose/phosphate buffer solution was 3.0.

The solution was filtered through a 0.22-micron filter and was useddirectly to dilute the protein solution.

Dilution of rhGDF-5 Solution with Trehalose Solution:

Bulk rhGDF-5 solution was dialyzed against phosphate buffer over nightusing a 3000 M.W. cut-off membrane at 2-8° C. After dialysis thesolution was slightly concentrated to 3.8 mg/ml. 14.47 ml of rhGDF-5solution was carefully transferred to a polypropylene flask, to whichtrehalose/phosphate buffer solution was slowly added to a final volumeof 100 ml while swirling the bottle. The solution was swirledoccasionally at room temperature for 15 minutes; the pH was measured andfound to be 3.0. Based on the UV reading, more trehalose/phosphatebuffer solution was added to obtain the desired protein concentration of0.5 mg/ml in 110 ml of solution; the pH was measured and found to be3.0; the UV reading indicated 0.50 mg/ml protein concentration. Thesolution was filtered through a 0.22-micron filter and was used directlyto dispense into vials.

Filling vials: 1.1 ml of rhGDF-5/trehalose solution was dispensedmanually into 5 ml Type 1 flint glass vials, and each vial was partlyclosed with a stopper prior to loading into the lyophilizer. Afterlyophilization, the stoppers were pressed and crimped. The product wasobtained as white to off-white cake.

TABLE 9 Stability of rhGDF-5 (0.5 mg/ml) plus trehalose (50 mg/ml) inphosphate buffer at pH 3.0 (Example 9) Solution Protein appearance mainpeak % Time and Cake after % recovery aggregates Temperature appearancereconstitution HPLC HPLC Time = zero Solid, white- clear 100 0 tooff-white, 1 month, Solid, white clear 100 0 50 C. to off-white 2 month,Solid, white clear 99.8 0 50 C. to off-white 3 month, Solid, white clear100 0 50 C. to off-white 1 month, Solid, white clear 100 0 250 C. tooff-white 2 month, Solid, white clear 98.7 0 250 C. to off-white 3month, Solid, white clear 99.7 0 250 C. to off-white 1 month, Solid,white clear 98.7 0 400 C. to off-white 2 month, Solid, white clear 97.40 400 C. to off-white 3 month, Solid, white clear 97.4 0 400 C. tooff-white

Example 10

Morselized collagen cylinder with 2.5 mg rhGDF-5 and 250 mg trehalose

Preparation of Trehalose Solution:

9.56 g of trehalose dihydrate was carefully weighed and transferred intoa sterile polypropylene bottle, to which 145 ml of purified water wasadded at room temperature and stirred slowly until a clear solution wasobtained. The clear trehalose solution pH was measured and found to be5.3. The volume was adjusted with purified water to obtain 150 ml finalvolume. The pH of the solution was measured and found to be 5.3. Thesolution was used directly to dilute the protein solution.

Dilution of rhGDF-5 Solution with Trehalose Solution:

16.45 ml of rhGDF-5 solution was carefully transferred to apolypropylene flask, to which trehalose solution was added carefully toadjust the volume to 120 ml; the pH was measured and found to be 2.9.The solution was stirred for 15 minutes at room temperature. The UVextinction coefficient was obtained to accurately calculate the proteinconcentration. Based on the UV reading, more trehalose solution wasadded to obtain the desired protein concentration of 0.5 mg/ml in 125 mlof solution; the pH was measured and found to be 2.9; the UV readingindicated 0.498 mg/ml protein concentration.

Dosing of Morselized Collagen Cylinders with rhGDF-5/Trehalose Solution

The solution was filtered through a 0.22-micron filter and the solutionwas used directly to dispense on pre-formed morselized collagencylinders that were packed in a Teflon mold. Each cylinder was dosedwith 5 ml of rhGDF-5/trehalose solution prior to lyophilization.

TABLE 10 Stability of morselized collagen cylinder with rhGDF-5 (2.5 mg)and trehalose (250 mg) per cylinder at 2-8° C. (Example 10) Pa- ram- 0 13 6 9 12 Test eter months month months months months months RP- % 87.2887.29 86.67 90.01 Study in progress HPLC main peak RP- % 0.0 0 0 0 HPLCag- gre- gates

The data below in table 11 show that the rhGDF-5 without any excipientsand deposited onto Healos® and lyophilized is stable at −20° C., but notat 2-8° C., as evidenced by the appearance of a late eluting peak in theRP-HPLC test of the 2-8° C. samples, but not the −20° C. samples.

TABLE 11 Stability of Healos ® strip with 5 ml of rhGDF-5 (0.5 mg/ml)without excipients at 2-8° C. and at −20° C. Parameter: % Late Eluting 01 2 6 9 12 18 Test Peak months month months months months months monthsRP-  2-8° C. 0 9.0 12.8 36.5 45.9 49.0 42.1 HPLC RP- −20° C. 0 2.6 0 1.62.2 2.7 3.8 HPLC

Different examples of making flowable morselized collagen/rhGDF-5 withexcipients and soluble collagen gel have been developed, and eachexample was evaluated for its performance, stability, and ease ofmanufacturing.

Morselized Collagen Example 1

Morselized collagen cylinder & rhGDF-5 lyophilized in the dry form

Collagen gel in wet form is kept separate

Both are kept separately in separate syringes at 2-8° C.

Both are mixed prior to injection

Morselized Collagen Example 2

Morselized collagen cylinder & rhGDF-5 & collagen gel mixed together inwet form (not lyophilized)

All are kept in a single syringe in wet form at 2-8° C.; ready to use

Morselized Collagen Example 3

Morselized collagen cylinder & rhGDF-5 & collagen gel lyophilized in thedry form

all are kept in dry form in a single syringe at 2-8° C.

rehydrate with water prior to injection

Morselized Collagen Example 4

Morselized collagen cylinder & collagen gel together as a paste

rhGDF-5 is kept separate in dry form

Both are kept separately in separate syringes at 2-8° C.

Both are mixed prior to injection

Morselized Collagen Example 5

Morselized collagen cylinder & collagen gel together in dry form

rhGDF-5 is kept separate in dry form

Both are kept separately in separate syringes at 2-8° C.

Reconstitute the rhGDF-5 with sterile water or bone marrow aspirate

Dry morselized collagen and collagen are mixed with reconstitutedrhGDF-5 solution prior to injection

The stability of rhGDF-5 was assessed using the following techniques:RP-HPLC, differential scanning calorimetry (DSC), circular dichroism(CD), polarized light microscopy (PLM), and also bioassay, with severalexcipients such as mannitol, sucrose, and trehalose in the presence andabsence of buffers and anti-oxidants. Several sucrose-containinglyophilized formulations of rhGDF-5 developed an undesirable yellowcolor and glassy cake structure during storage and therefore were notpromising.

The melting behavior of lyophilized rhGDF-5 formulations was studiedusing DSC. The DSC data demonstrated that both trehalose andmannitol-based formulations significantly improved the thermal stabilityof bulk rhGDF-5.

FIGS. 1, 2 and 3 show a comparison of the DSC profiles of the trehaloseformulation and mannitol formulation of rhGDF-5 compared to that of bulkrhGDF-5. Bulk rhGDF-5 displays two major transitions: one near 40° C.and the other near 85° C. The high temperature transition probablyrepresents the protein's thermal unfolding. It is interesting to notethat the melting temperature (T_(m)) of the first endothermic transitionis increased by 7-14° C. in the presence of excipients. When consideredby itself, this study suggests that both trehalose and mannitol could beequally effective as a stabilizer.

PLM (polarized light microscopy) of the trehalose/rhGDF-5 formulation isshown in FIG. 4. The sample does not show a major birefringencephenomenon. Thus, the system is amorphous, which is ideal fortherapeutic applications. FIG. 5 shows the PLM of the mannitol/rhGDF-5formulation after a period of storage. Many crystals were observed inthe sample, indicating that the mannitol had crystallized duringstorage. This result suggests that trehalose is the better lyoprotectantfor rhGDF-5. The far UV CD spectra revealed that trehalose-basedformulations have a secondary structural distribution comparable to thatof native bulk protein.

Real time stability studies by RP-HPLC of lyophilized rhGDF-5 withvarious excipients clearly demonstrated that rhGDF-5 in the presence oftrehalose, at either 50 mg/ml or 100 mg/ml concentrations, with orwithout buffers, and with or without polysorbate, consistently impartedimproved stability upon rhGDF-5 at both 2-8° C. and 15-25° C. storageconditions, whereas mannitol failed to provide the same level ofstability under similar storage conditions.

The real time stability studies of lyophilized cake formulations clearlyshowed that mannitol did not stabilize the protein, as evidenced by themain peak being decreased significantly while the aggregate peak isincreased at room temperature, as well as 2-8° C. storage conditions.The aggregates are the most undesirable species in the proteinformulations as they may cause immunological reactions and side effects.In contrast, trehalose stabilized the protein very well by inhibitingthe formation of aggregates and protecting the main peak, particularlyat 2-8° C. storage conditions, as evidenced in real time stabilitystudies. Thus trehalose is better than mannitol in stabilizing rhGDF-5in formulations. Also, the real time stability data indicate thatrhGDF-5/trehalose formulations having phosphate or glycine as a bufferto control the pH is even better than rhGDF-5/trehalose formulationswithout buffers. The real time stability data indicate that the idealstorage of rhGDF-5 trehalose/glycine formulations is at 2-8° C., andalso that storage at 25° C. is adequate.

In addition to the favorable biochemical and biophysical data oftrehalose-based rhGDF-5 formulations, these formulations also showedpotency in the alkaline phosphatase biological assay. Physical chemicalmethods of analysis, in vitro assays, and real time stability data showthe promise of trehalose as a superior excipient in stabilizing rhGDF-5in a lyophilized stand-alone product, as well as collagen-basedcombination products for use in the treatment of a variety ofmusculoskeletal disorders.

Example 11 Solubility of rhGDF-5 in Different Ionic Strength Solutionsand Two pH Buffers (pH 3 and pH 4)

Various ionic strength solutions of sodium phosphate buffer were used inthis study. A bulk protein solution was concentrated to approximately 10mg/mL and dialyzed with 5, 10, 25, 50 and 100 mM phosphate buffers at pH3 or pH 4. After dialysis, the samples were checked for clarity andanalyzed for protein concentration on an UV-Vis spectrophotometer.Detailed procedures are described below.

Buffer Preparations

100 mM Phosphate Buffer at pH 3:

13.5 mL of concentrated H₃PO₄ (14.8 M) solution was transferred to a2000-mL beaker, to which DI water was added up to 1900-mL mark. Thesolution was titrated with a NaOH solution to pH 3 and transferred to a2000-mL graduated cylinder. Additional water was added to make up 2000mL. The content was transferred back to the beaker and mixed thoroughly.

50 mM Phosphate Buffer at pH 3:

6.76 mL of concentrated H₃PO₄ (14.8 M) solution was transferred to a2000-mL beaker, to which DI water was added up to 1900-mL mark. Thesolution was titrated with a NaOH solution to pH 3 and transferred to a2000-mL graduated cylinder. Additional water was added to make up 2000mL. The content was transferred back to the beaker and mixed thoroughly.

25 mM Phosphate Buffer at pH 3:

3.39 mL of concentrated H3PO4 (14.8 M) solution was transferred to a2000-mL beaker followed by addition of DI water to 1900-mL mark. Thesolution was titrated with a NaOH solution to pH 3 and transferred to a2000-mL graduated cylinder. Additional water was added to make up 2000mL. The content was transferred back to the beaker and mixed thoroughly.

10 mM Phosphate Buffer at pH 3:

1.35 mL of concentrated H₃PO₄ (14.8 M) solution was transferred to a2000-mL beaker to which DI water was added up to 1900-mL mark. Thesolution was titrated with a NaOH solution to pH 3 and transferred to a2000-mL graduated cylinder. Additional water was added to make up 2000mL. The content was transferred back to the beaker and mixed thoroughly.

5 mM Phosphate buffer at pH 3:

0.676 mL of concentrated H₃PO₄ (14.8 M) was transferred to a 2000-mLbeaker followed by addition of DI water to 1900-mL mark. The solutionwas titrated with a NaOH solution to pH 3 and transferred to a 2000-mLgraduated cylinder. Additional water was added to make up 2000 mL. Thecontent was transferred back to the beaker and mixed thoroughly.

Sample Preparation

Bulk protein rhGDF-5 (Lot #2142131) was thawed at 2-8° C. The bulkprotein solution (24 mL at 3.8 mg/mL) was concentrated using acentrifugal filtration device (Pall Life Science, Cat #OD010C37, 10KMWCO) to a volume of approximately 6 mL. Approximately 0.9 mL of theconcentrated rhGDF-5 solution was transferred to each dialysis cassette(Pierce, Cat #66380) and dialyzed against the phosphate buffers overnight at room temperature. The concentrated rhGDF-5 solutions werecarefully removed from the dialysis cassettes and placed in small glassvials to check solution clarity. Protein concentrations were determinedon an UV-Vis spectrophotometer as described in the Analytical Methodssection.

Solubility at pH 4

Buffers of pH 4.0 were prepared from the pH 3 buffers by adding moreNaOH solution to the pH 3 buffers. The protein solutions were dialyzedagainst the pH 4 buffers at room temperature over night. The sampleswere analyzed for solution clarity and protein concentration.

Analytical Methods

Solution samples in small glass vials were checked for clarity andparticles. The sample vials were inspected using a vertical lightagainst a black background. The clarity of the test samples was comparedwith a pure water sample as a control. The pH of each solution samplewas measured directly using a calibrated pH meter.

Results

The results of the solubility study of 10 mg/ml rhGDF-5 solutions showedthat the lower ionic strength buffers of sodium phosphate at 5, 10, and25 mM yielded clear solutions, indicating good solubility, while higherionic strength buffers of sodium phosphate at 50 and 100 mM yielded hazysolutions, indicating poor solubility. At pH 4, the 5 and 10 mM sodiumphosphate buffers yielded hazy solutions, indicating poor solubility.Sodium phosphate buffers at 25, 50 and 100 mM yielded clear solutionsafter centrifugation, but had nearly zero protein recovery, indicatingthat the protein had precipitated. Thus, low ionic strength buffers nearpH 3 would be preferable to higher ionic strength buffers at higher pH.

Example 12 Stability of rhGDF-5 at Various Temperatures in VariousBuffers with 5% Trehalose

In this study various buffers were tested for their effects onprotecting 0.7 mg/mL rhGDF-5 in a 5% trehalose solution duringlyophilization and storage at 5° C. The buffers tested were 5 mMglycine-HCl pH3, 5 mM sodium phosphate pH 3, 5 mM sodium citrate pH 3,10 mM sodium lactate pH 3, 0.01% TFA in water, 1 mM HCl, and a controlsolution of rhGDF-5 in 1 mM HCl with no trehalose present. The bufferswere prepared as follows:

5 mM Glycine Buffer, pH 3

A 2000-mL beaker was charged with 0.75 g of glycine (MW 75.05 g) and1900 ml of DI water; the solution was titrated with a HCl solution to pH3 while it was stirring. Additional water was added to make up 2000 mLand mixed thoroughly.

5 mM Citrate Buffer, pH 3

A 2000-mL beaker was charged with 2.11 g of citric acid monohydrate(MW210.14) and 1900 ml of DI water; the solution was titrated with aNaOH solution to pH 3. Additional water was added to make up 2000 mL andthe solution was mixed.

5 mM Phosphate Buffer, pH 3

0.676 mL phosphoric acid solution (14.8M) was transferred to a 2000-mLbeaker containing 1900 mL of DI water; the solution was titrated with aNaOH solution to pH 3. Additional water was added to make up 2000 mL andthe solution was mixed thoroughly.

10 mM Lactate Buffer, pH 3

A 2000-ml size beaker was charged with 1.81 g lactic acid (MW 90.08) and1900 ml of DI water; the resulted solution was titrated with a NaOHsolution to pH 3. Additional water was added to make 2000 mL and thesolution was mixed thoroughly.

1 mM HCl Solution

1 mL of 2N HCl solution was transferred to a 2000-mL beaker containing1900 ml of DI water. Final volume of the solution was adjusted to 2000mL mark by adding more DI water.

0.01% TFA Solution

0.2 mL TFA solution was transferred to a 2000-mL beaker containing1900ml of DI water. Final volume of the solution was adjusted to 2000 mL byadding additional water and the solution was mixed thoroughly.

Formulation Preparation

Bulk protein rhGDF-5 (Lot #2142131) was thawed at 2-8° C. The bulkprotein solution (55 mL at 3.8 mg/mL) was concentrated using acentrifugal filtration device (Pall Life Science, Cat #OD010C37, 10KMWCO) to a volume of approximately 10 mL. Approximately 1.4 mL ofconcentrated rhGDF-5 solution was transferred to each dialysis cassette(Pierce, Cat #66380) and the cassettes were dialyzed against the testbuffers over night at 2-8° C.

The rhGDF-5 solutions were removed carefully from the dialysis cassettesand transferred to small glass bottles. Protein concentrations of thesolutions were measured using an UV-Vis spectrophotometer. The proteinwas formulated at approximately 0.7 mg/mL with 5% (w/v) trehalose in thetest buffers and filtered through 0.22 μm filters. The solutions werestored at 2-8° C. prior to lyophilization.

Filling and Lyophilization

Each formulated solution was filled into 3-mL glass vials (WestPharmaceutical Services, Cat #68000316) at 1 mL/vial. The vials wereclose partially with stoppers (West Pharmaceutical Services, Cat#99150630) and transferred to the lyophilizer (FTS System, LyoStar II).Thermocouples were placed in placebo vials to monitor the lyophilizationprocess. As a control, another formulation with no trehalose was alsotested. 200 μL of 4.5 mg/mL rhGDF-5 in 1 mM HCl solution was transferredto each glass vial and lyophilized.

Analytical Methods

Integrity of Lyophilization Cakes

The lyophilized sample was checked at each time point for cracks,shrinkage and collapse of lyophilized cakes.

Reconstitution Time

One milliliter of DI water was added to each lyophilized sample andmixed gently. The reconstitution time was recorded.

Solution Clarity—Visual Appearance

Solution samples in small glass vials were checked for clarity andparticles. The sample vials were inspected using a vertical lightagainst a black background. The clarity of the test samples was comparedwith a pure water sample as a control.

pH Method

pH of each solution sample was measured directly using a calibrated pHmeter.

UV Spectroscopy

Protein concentration was determined using an UV-Vis spectrophotometer.The concentration of rhGDF-5 was calculated using an extinctioncoefficient of 1.16 mL/mg*cm at 280 nm.

HPLC Method

The non-reduced rpHPLC method (TM 0051 D) was used to monitor modifiedspecies of the protein. The test samples were diluted with 50 mM aceticacid to approximately 0.1 mg rhGDF-5/mL solution. The diluted samples(50 μl each) were injected onto the HPLC column (Vydac 218TP52, C18column). The samples were eluted with 0.15% (v/v) TFA in water and 0.15%(v/v) TFA in acetonitrile as the mobile phase at 0.3 ml/min. The elutedpeaks were detected at 214 nm. Percentage of each peak area wascalculated to monitor the changes of the main peak and minor peaks(degraded peaks).

Size Exclusion Chromatography (SEC)

Protein aggregation was monitored using a SEC method. Typically, 30 μLof each test sample was injected directly onto the SEC column (TOSOHBioscience, Cat #08540) and eluted with 0.1% (v/v) TFA and 45% (v/v)acetonitrile in water at a rate of 0.5 ml/min. The protein peaks weremonitored at 280 nm and the percentage of aggregate was calculated.

Gel Electrophoresis

Protein aggregates and degraded small pieces were also monitored using agel electrophoresis method. Typically, approximately 10 μg protein wasdried and reconstituted with 70 μL of SDS-PAGE sample buffer(Invitrogen, Cat #LC2676) with or without 10% β-mercaptoethanol. Thesamples were incubated at 95° C. for 5 minutes. Approximately 18 μL ofeach sample was loaded on to gels (Invitrogen, Cat #NP0341 Box). Thegels were run using a running buffer (Invitrogen, Cat #NP0002) at 200voltages for about 35 minutes. The gels were then stained withSimplyblue solution (Invitrogen, Cat #LC6060) and de-stained with DIwater. The gels were scanned and images were collected.

Biological Activity Assay

Only the 6-month stability samples (glycine formulation and HClformulation) were analyzed for biological activity. The cell-based assay(TM 0046) was used to measure alkaline phosphatase activity to determinethe stability of the samples.

Water Content

The moisture content assay was conducted by PDD using a Karl FischerTitration method.

Results

Integrity of Lyophilization Cakes

Test sample cakes in all storage conditions appeared solid and white tooff-white from the time zero through the 9-month time point. Slightshrinkage was observed around the cakes, or the cakes were slightlyseparated from glass wall of the vials, as is commonly observed whensugars such as trehalose or sucrose are used as a bulking agent. Therewas no collapse of cake in all the test samples. Usually cake collapsemay alter the reconstitution time and lead to protein instability.White, fluffy and light cakes were obtained in the formulation with notrehalose present.

Reconstitution Time

One milliliter of water was added to each sample vial at the time oftesting. The sample was gently mixed and reconstitution time wasrecorded. Approximately 30 to 40 seconds were required for thecompletion of cake solubility.

Solution Clarity

Reconstituted solution samples were inspected under a vertical light ona black background; all sample solutions are found clear and colorless

pH

The pH of reconstituted solution was measured using a calibrated pHmeter. Through out the course of study there were no significant changesin pH value across all the formulations. pH of the formulation samplescontaining trehalose/buffers was around 3.0±0.2. The pH of theformulation without trehalose was about 4.0.

UV Spectroscopy

The protein concentration was measured on an UV-VIS spectrophotometer.Through out the study there were no significant changes in proteinconcentration in rhGDF-5/trehalose formulations containing the glycinebuffer, phosphate buffer, citrate buffer, lactate buffer, or 0.01% TFA.The absorbance at 280 nm was increased in the rhGDF-5/trehalose/HClformulation when it was stored at 25° C./60% and 40° C./75% RH. Theconcentration of protein appeared to be increasing in the formulationthat was stored at 40° C.; the initial protein concentration of 0.7mg/mL at time zero was increased to 1 mg/mL at the 6-month time point.This may imply that trehalose might degrade to furfural compounds, whichhave similar absorbance at 280 nm.

Non-Reduced rpHPLC Results

The non-reduced rpHPLC method was used to monitor the degradationspecies of rhGDF-5, which were formed by methionine oxidation,deamidation reaction and other reactions. There were no significantchanges in percentage of the main peak for all the formulations storedat 2-8° C. and 25° C. for 9 months, except for the HCl formulation andthe formulation without trehalose. Both formulations had less than 90%of the main peak at the 9-month time point.

However, when the formulations were stored at accelerated storageconditions such as 40° C./75% RH, only one formulation (i.e.rhGF-5/trehalose/glycine) had greater than 91% of the main peak at the6-month time point. The other formulations were not as stable asrhGDF-5/trehalose/glycine formulation under the accelerated storageconditions. Particularly, rhGDF-5/trehalose/HCl formulation had only 66%of the main peak at the 6-month time point. FIGS. 6 and 7 shows the HPLCchromatograms of rhGDF-5/trehalose/glycine formulation andrhGDF-5/trehalose/HCl formulation stored at 40° C./75% RH for 6 months.FIGS. 8, 9, and 10 show the protein recovery of the various bufferstested at storage at 5°, 25°, and 40° C.

The results from rpHPLC analysis indicate that a combination oftrehalose and glycine buffer provides the best stability to lyophilizedrhGDF-5 during the storage. Additionally, the formulation ofrhGDF-5/trehalose/HCl is less stable because the strong acid of HCl mayhave some destabilizing effects on both protein as well as trehalose.

Example 13 Stability of rhGDF-5 at Various Temperatures in a pH 3Glycine Buffer with 5% Trehalose

In this study, rhGDF-5 was formulated at approximately 0.01, 0.03, 0.1,2.5, 4.5 and 9 mg/mL with 5% (w/v) trehalose and 5 mM glycine-HCl bufferat pH 3. The formulated solutions were used to fill in 3-mL glass vialsat 1 mL/vial and the vials were lyophilized. The lyophilized samplevials were stored at 2-8° C., 25° C./60% RH and 40° C./75% RH. At eachdesignated time point, the samples were analyzed for the stability ofthe products. The methods used in this study include cake appearance,reconstitution time, solution clarity, pH, rpHPLC (reverse phase highperformance liquid chromatography), UV (ultra-violet spectroscopy), SEC(size exclusion chromatography) and gel electrophoresis. After 6-monthstorage at the three storage conditions, it was found that there were nosignificant changes observed in the formulations with proteinconcentrations from as low as 0.1 mg/ml to as high as 9 mg/mL. When theprotein concentration was too low, such as 0.01 and 0.03 mg/ml, theexisting methods were not robust enough to detect minor changes.

The results of this study indicate that lyophilized rhGDF-5 formulationscontaining trehalose and glycine-HCl with varying protein concentrationswere stable at 2-8° C., 25° C./60% RH for at least 6 months. Slightchanges in rpHPLC profile were seen in the product stored at acceleratedstorage conditions of 40° C./75% RH at the 6-month time point.

Example 14 Stability of Different Concentrations of rhGDF-5 at VariousTemperatures in a pH 3 Glycine Buffer with 5% Trehalose

In this study rhGDF-5 was formulated with 5% (v/w) trehalose and 5 mMglycine buffer at pH 3 with concentrations of rhGDF-5 of 0.01, 0.03,0.1, 2.5, 4.5, and 9.0 mg/ml. Additionally, one formulation of 4.5 mg/mlrhGDF-5 was prepared with 10% (w/v) trehalose and 5 mM glycine buffer(pH 3) for comparison. The formulated solutions were then filled in 3-mLglass vials at 1 mL/vial and lyophilized. The lyophilized samples werestored in stability chambers.

5 mM Glycine-HCl Buffer, pH 3

3×0.75 g glycine (MW 75.07 g) was weighed into 3×2000-mL beakers andapproximately 1900 mL of DI water was added to each beaker. Thesolutions were titrated with a HCl solution to pH 3. Additional waterwas added to the final volume of 2000 mL for each beaker and mixedthoroughly.

Formulation Preparation

Bulk protein rhGDF-5 (Lot #2142131) was thawed at 2-8° C. The proteinsolution (96 mL at 3.8 mg/mL) was concentrated using 4 centrifugalfiltration devices (Pall Life Science, Cat #OD010C37, 10K MWCO) to atotal volume combined of approximately 24 mL. Approximately 3×8 mL ofthe concentrated rhGDF-5 solution was transferred to 3× dialysiscassettes (Pierce, Cat #66380) and dialyzed against the glycine-HClbuffer over night at 2-8° C.

The rhGDF-5 solutions were transferred from the dialysis cassettes to asmall glass bottle. Protein concentration was measured using an UV-Visspectrophotometer. The protein was formulated at various concentrationswith 5 or 10% (w/v) trehalose and 5 mM glycine buffer as describedabove. The formulated solutions were filtered with 0.22 μm filters andstored at 2-8° C. prior to lyophilization.

Fill and Lyophilization

Each of the formulated solutions was filled into 3-mL glass vials (WestPharmaceutical Services, Cat #68000316) at 1 mL/vial. Stoppers (WestPharmaceutical Services, Cat #99150630) were partially placed on thevials. The sample vials were transferred to the lyophilizer (FTS System,LyoStar II). Thermocouples were placed in placebo vials to monitor thetemperature profiles during lyophilization process.

Analytical methods used were similar to those described above inexamples 11 and 12.

Results

Integrity of Lyophilization Cakes

Test sample cakes in all storage conditions appeared solid and whitefrom time zero through 6-month time point. Slight shrinkage was observedaround the cakes or the cakes were slightly separated from glass wall ofthe vials. This is quite common when sugars, such as trehalose orsucrose are used as a bulking agent. No collapsed cakes were seen in allthe test samples.

Reconstitution Time

One milliliter of water was added to each sample vial at the time pointsof testing. The vial was gently mixed and reconstitution time wasrecorded. It took approximately 30 to 40 seconds for the cake to go intosolution.

Solution Clarity

All reconstituted samples appeared clear and colorless when the proteinsolutions were inspected with a vertical light against a blackbackground.

pH

The reconstituted solution was used to measure pH. No significantchanges in pH were observed in all the samples through the course of thestudy. The pH values of the formulations were in the range of 3.0 to3.3.

UV Spectroscopy

The protein concentration was measured using the UV spectroscopy method.The UV spectrum could also provide information on protein aggregation(baseline light scattering). For protein concentrations from 0.01 to 0.1mg/mL, a 10-mm cuvette was used. For protein concentrations from 2 to 9mg/mL, a 1-mm cuvette was used with no dilution or no sample disrupted.No significant changes in protein concentrations were observed in thesamples of 0.1 to 9 mg/mL through out the course of the stability study.For the low concentration samples of 0.01 and 0.03 mg/ml, more variationwas seen because the absorbance was too low. A new sample preparationmethod should be needed for the low concentration samples for futurestudies.

Non-Reduced rpHPLC Results

The non-reduced rpHPLC is used to monitor degraded species of rhGDF-5,such as methionine oxidation and deamidation. No significant changes inpercentage of the main peak were observed in all the samples stored at2-8° C., 25° C. and 40° C. through out 6-month storage. The main peak ofrhGDF-5 of samples that were stored for 6-months was still recoveredwith ≧96% and it was comparable to the data obtained from time zerosamples. The low concentration samples of 0.01 and 0.03 mg/mL weredifficult to analyze by the HPLC method. A new sample preparation shouldbe needed for future studies.

SEC

SEC was used to monitor protein aggregation. There were no significantchanges found in aggregation of all the samples, which were testedthroughout the 6-month stability study. The low concentration samples of0.01 and 0.03 mg/mL were not analyzed.

Gel Electrophoresis

Protein aggregation and degradation species were also monitored usinggel electrophoresis. There were no significant changes found in all thesamples through out 6-month storage.

Small fragments of the protein were not formed in any sample during thestorages, as these were not found on reduced SDS-PAGE

Water Content

The water contents of the samples were low, ranging from 0.19 to 0.32%.No correlation or trend was seen between the protein concentrations andwater contents.

The results indicate that the lyophilized rhGDF-5 products in thepresence of trehalose and glycine buffer are stable at 2-8° C., 25°C./60% RH and 40° C./75% RH for at least 6 months, as evidenced byrpHPLC and SEC chromatography. The protein can be formulated at variousconcentrations ranging from 0.1 to 9 mg/mL (pre-lyophilization) with 5%(w/v) trehalose/5 mM glycine-HCl buffer (pH 3) and lyophilized. When theprotein was formulated at low concentration such as 0.01 mg/mL and 0.03mg/mL, the existing methods have some limitations to detect the changes.

The present invention has been described relative to illustrativeembodiments. Since certain changes may be made in the above formulationswithout departing from the scope of the invention, it is intended thatall matter contained in the above description or shown in theaccompanying drawings be interpreted as illustrative and not in alimiting sense. For example, one skilled in the art will recognize thatthe formulation of the illustrative embodiments of the invention is notlimited to use with BMP and can be used with other biomolecules for anysuitable biologic system.

It is also to be understood that the following claims are to cover allgeneric and specific features of the invention described herein, and allstatements of the scope of the invention, which, as a matter oflanguage, might be said to fall there between.

What is claimed is:
 1. A method of stabilizing a BMP comprising thesteps of: a.) providing a composition containing at least one BMP,wherein the at least one BMP is rhGDF-5, and an amount of trehalosesufficient to stabilize said BMP, and b.) adding at least one excipientselected from the group consisting of glycine, polysorbate 80,polysorbate 20, and mixtures thereof, wherein said excipient has a pH ofabout 2.5 to about 3.5, and c.) lyophilizing the composition.